HAY, JORDAN O.*, MIGUEL PINEROS, LEON V. KOCHIAN, AND ROGER M. SPANSWICK. Department of Plant Biology, U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, NY 14853. - Isolation of rice aleurone protoplasts and whole cell patch clamping.
Very little is known about the physiology of sucrose transport into
developing rice grains. While the post-phloem unloading sucrose
transport pathway has been described in detail, quantitative studies
of sucrose uptake including the role of membrane transport are
lacking. This has limited our understanding of carbon partitioning and
the control of rice yield. We are interested in studying the
sucrose-proton cotransport system that has been reported as being
expressed in rice endosperm. Based on previous reports that the
aleurone layer is symplastically isolated from the phloem, we chose to
investigate sucrose-proton cotransport in rice aleurone cells.
Aleurone protoplasts were isolated from Oryza sativa aleurone
peels using standard cell wall degradation enzymes. Very high
osmolarity, 0.8-1 M mannitol or sorbitol, was essential for obtaining
high yields of 5,000-30,000 viable protoplasts/10 grains. Protoplasts
were 20-40 mm in diameter and had granular
cytoplasm. These traits were consistent with previous reports on rice
aleurone layer morphology and were confirmed after our study of grain
anatomy using light microscopy. The aleurone protoplasts proved to be
a system amenable to whole cell patch clamping. We formed high
resistance seals (3 Giga-ohms) without difficulty and assessed whole
cell currents from protoplasts bathed in solutions containing
different levels of K+. The whole cell conductance of these
cells was dominated by an instantaneous outward current as well as a
slow developing, time dependent inward current. Future experiments,
designed for modeling rice yield, include testing the suitability of
this aleurone whole cell system for quantitative studies of
sucrose-proton cotransport.
Key words: aleurone, carbon partitioning, modeling, patch clamping, rice, sucrose transport